Gaining a better understanding of the mechanisms that govern cartilage structure is essential to predict its biomechanical properties, particularly its load-bearing and lubricating abilities. This knowledge is also a prerequisite for the success of tissue engineering and regenerative medicine strategies to grow, repair, and reintegrate cartilage. Cartilage functional properties are influenced by biochemical and microstructural changes occurring in development, degeneration, aging and diseases. To study cartilage physical properties (e.g., osmotic swelling properties and hydration) an array of techniques is required that probe not only a wide range of length scales but also statistically representative volumes of the sample. The approach of controlled hydration provides a direct means of determining functional properties of cartilage and of other tissues. Specifically, we have used controlled hydration of cartilage to measure physical/chemical properties of the collagen network and of the proteoglycans (PG) independently within the extracellular matrix. This approach entailed modeling the cartilage tissue matrix as a composite material consisting of two distinct phases: a collagen network and a concentrated PG solution trapped within it. In these earlier pilot studies, we used this approach to determine pressure-volume curves for the collagen network and PG phases in native and in trypsin-treated normal human cartilage specimen, as well as in cartilage specimen from osteoarthritic (OA) joints. In both normal and trypsin-treated specimens, collagen network stiffness appeared unchanged, whereas in the OA specimen, collagen network stiffness decreased. Our findings highlighted the role of the collagen network in limiting normal cartilage hydration, and in ensuring a high PG concentration, and thus, swelling pressure within the matrix, both of which are essential for effective load bearing in cartilage and joint lubrication, but are lost in OA. A shortcoming of this approach was that it required excised tissue slices to obtain these osmotic titration curves. This lead to long equilibration times requiring several person-days to study a single cartilage specimen, making this approach unsuitable for routine pathological analysis or in tissue engineering applications. Subsequently, we designed and built a new tissue micro-osmometer to perform these experiments practically and rapidly (US Patent No. 7,380,477). This instrument can measure minute amounts of water absorbed by small tissue samples (< 1 microgram) as a function of the equilibrium activity (pressure) of the surrounding water vapor. A quartz crystal sensitively and precisely detects the water uptake of the tissue specimen attached to its surface. Varying the equilibrium vapor pressure surrounding the specimen induces controlled changes in the osmotic pressure of the tissue layer. We used the tissue micro-osmometer to obtain a profile of the osmotic compressibility or stiffness of cartilage specimens as a function of depth from the articular surface to the bone interface. The apparatus also allows us to assess the mechanical integrity of developing tissues and osmotic compatibility of tissue-engineered cartilage (or ECM), which is essential for improving integration and viability following implantation in regenerative medicine applications. We have developed an experimental procedure for mapping the local elastic properties of cartilage using the atomic force microscope (AFM). This technique utilizes the precise scanning capabilities of AFM to generate large volumes of compliance data from which we extract the relevant elastic properties. We mapped the osmotic modulus of bovine cartilage samples by combining tissue micro-osmometry with force-deformation measurements made by the AFM. Knowledge of the local osmotic properties of cartilage is particularly important since the osmotic modulus defines the compressive resistance to external load. We found that the water retention is stronger in the upper and deep zones of cartilage, where collagen fibers are orderly organized, than in the middle zone where they are randomly arranged. We have constructed the elastic and osmotic modulus maps for the different layers. The latter that is a combination of the elastic and swelling properties, exhibits much stronger spatial variation reflecting the highly heterogeneous character of the tissue. We correlated the mechanical measurements with observations made by MRI imaging on the same cartilage specimens. This approach has the potential to develop novel diagnostic methods. A major objective of tissue engineering is to mimic the ECM environment. However, the complexity of interactions between ECM and cells makes it difficult to design materials for regenerative medicine applications. Previous studies have indicated that the chemical structure of the scaffold is critical. Molecular factors (e.g., hydrophilic or hydrophobic character of the scaffold, stiffness, charge density of the polymer) significantly influence cell adhesion, spreading and growth. In collaboration with researchers at the Carnegie Mellon University we developed novel nanostructured hydrogels, which have potential as an artificial ECM, which can act as a macroscopic scaffold for tissue regeneration. Experimental results obtained by macroscopic (osmotic swelling pressure measurements) and microscopic techniques (SANS, SAXS, DLS) aimed a quantifying the interactions between the main macromolecular components of the ECM, yield insights both into the properties of aggrecan assemblies at a supramolecular level and into the mechanism of load bearing of cartilage. Measurements made on aggrecan, hyaluronic acid (HA), and aggrecan/HA solutions indicate that the osmotic pressure, molecular organization, and dynamic response of PG assemblies are governed by the bottlebrush-shaped aggrecan molecule. Osmotic pressure measurements allow us to quantify the contributions of individual components of ECM (e.g., aggrecan, HA, and collagen) to the total swelling pressure. Our osmotic pressure measurements on aggrecan/HA systems showed evidence of self-assembly of the bottlebrush shaped aggrecan subunits into microgel-like assemblies. Complexation with HA enhances aggrecan assembly. The osmotic pressure of aggrecan-HA complexes decreases with decreasing the ratio of aggrecan to HA. SANS reveals that there is no significant interpenetration between neighboring aggrecan molecules. Neither osmotic pressure measurements nor SANS indicate significant interactions between the collagen network and the aggrecan bottlebrushes. We are developing a multiscale experimental approach to study ECM in which quantitative MR imaging is combined with chemical composition mapping (HDIR spectroscopy) and high resolution mechanical measurements made by the AFM. Our pilot study made on 2.5 year-old bovine cartilage yields consistent results between the NMR parameters, the chemical composition and structure of the ECM, and its mechanical properties. HDIR validates the use of MR imaging of tissue composition. AFM bridges MR and HDIR by establishing relationships between chemical composition, structure and mechanical tissue function. Our hope is to identify and quantify molecular and microstructural changes that allow us to monitor early changes in the tissue that may later lead to cartilage degeneration. This research activity was supported by the NICHD DIR Director's Award. Collectively, these quantitative physical/chemical approaches are helping us get closer to understanding the basis of ECM's functional properties in general, and cartilage in particular, their changes in development, as well as their loss in diseases, degeneration and aging.
